Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms and are responsible for the supply of monomeric precursors required for DNA replication and DNA repair. The central role of RNRs in nucleic acid metabolism has made them the successful target of at least three drugs used clinically in the treatment of cancers, hydroxyurea, gemcitabine, and clofarabine, as well as the clinical drug candidate, triapine. The RNRs serve as a paradigm for understanding the exquisite control of reactions involving stable and transient protein- and nucleotide-based radical intermediates. The class Ia enzymes also serve as the paradigm for an unprecedented mechanism of radical propagation over a distance of >35 , involving the transient formation of aromatic amino acid radical intermediates (Y or W) by proton-coupled electron transfer (PCET). The present proposal is focused on the class Ia RNRs from E. coli and human. Our ability to incorporate unnatural amino acids (UAAs) site-specifically into the two subunits (? and ?) of the E. coli RNR, coupled with the tools of high field EPR and ENDOR spectroscopies, stopped-flow kinetics, structure, and computation, will allow us to determine if the PCET process occurs with proton transfer that is orthogonal or co-linear to the proposed ET pathway. The UAAs 3-nitrotyrosine (NO2Y) and 3- aminotyrosine (NH2Y) serve as probes of the ground state and intermediate states, respectively, of E. coli RNR, and are sensitive to changes in protein conformation, hydrogen bonding networks, and redox potentials. A method for the site-specific incorporation of the UAA 2,3,5-trifluorotyrosine (F3Y) into ? will be developed, and the pH rate profile of the resulting mutant-?s will be measured to test the proposed differences in PCET mechanism between the two subunits. Humans have two RNRs: ?/?, involved in DNA replication, and ?/?', involved in DNA repair and mitochondrial DNA replication. Basic biochemical studies of these two RNRs will be carried out to understand (a) the stability of the radical initiation cofactor, (b) the ability of ?' to function as a catalase, (c) the active quaternary structures of ?/? and ?/?', and (d) the similarities or differences in the PCET pathway compared to the E. coli enzyme. PUBLIC HEALTH RELEVANCE: Ribonucleotide reductases (RNRs) are enzymes responsible for the conversion of nucleotides to deoxynucleotides, the monomeric building blocks of DNA. Thus, they play a key role in regulating DNA replication and repair, and are a successful clinical target for several types of cancer. The aim of this proposal is to study RNR's unique mechanism, the understanding of which will aid in developing novel RNR inhibitors.